Cannabidiol Protects Dopaminergic-like Neurons against Paraquat- and Maneb-Induced Cell Death through Safeguarding DJ-1CYS106 and Caspase 3 Independently of Cannabinoid Receptors: Relevance in Parkinson's Disease.

Parkinson's disease (PD), a progressive neurodegenerative movement disorder, has reached pandemic status worldwide. This neurologic disorder is caused primarily by the specific deterioration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc). Unfortunately, there are no therapeutic agents that slow or delay the disease progression. Herein, menstrual stromal cell-derived dopamine-like neurons (DALNs) intoxicated with paraquat (PQ2+)/maneb (MB) were used as a model system to elucidate the mechanism by which CBD protects the neural cell from apoptosis in vitro. According to immunofluorescence microscopy, flow cytometry, cell-free assay, and molecular docking analysis, we demonstrate that CBD offers protection to DALNs against PQ2+ (1 mM)/MB (50 µM)-induced oxidative stress (OS) by simultaneously (i) decreasing reactive oxygen species (ROS: O2•-, H2O2), (ii) maintaining the mitochondrial membrane potential (ΔΨm), (iii) directly binding to stress sensor protein DJ-1, thereby blunting its oxidation from DJ-1CYS106-SH into DJ-1CYS106-SO3, and (iv) directly binding to pro-apoptotic protease protein caspase 3 (CASP3), thereby disengaging neuronal dismantling. Furthermore, the protective effect of CBD on DJ-1 and CASP3 was independent of CB1 and CB2 receptor signaling. CBD also re-established the Ca2+ influx in DALNs as a response to dopamine (DA) stimuli under PQ2+/MB exposure. Because of its powerful antioxidant and antiapoptotic effects, CBD offers potential therapeutic utility in the treatment of PD.

Multi-generational effects under single and pulse exposure scenarios in two monophyletic Daphnia species.

Anthropogenic activities commonly relate to a set of diffuse and point contamination sources, from industrial, domestic or agricultural outputs, characterized by a chemical cocktail exposure and consequent disturbances of natural ecosystems. Different species may present different sensitivities to contaminants, even when phylogenetically close. This study used two monophyletic Daphnia species from tropical and temperate environments, Daphnia similis and Daphnia magna respectively, to evaluate the variation of their sensitivity to Pb (if any) and fitness during a multi-generational exposure and recovery. To accomplish that, standard acute immobilization tests were done on specific generations. Tests were carried out with exposures to 1) potassium dichromate (K2Cr2O7) to evaluate organisms' sensitivity/fitness, 2) Pb, to monitor variation on Pb sensitivity and 3) the fungicide mancozeb, providing a pulse toxicity approach on generational Pb acclimated daphnids. Since growth is an important trait related to organisms' fitness, organisms' size measurements were also monitored. In addition, organisms were maintained under two different dietary regimes. Our results indicate no variation on daphnids sensitivity to K2Cr2O7, except for D. similis from a recovery period under food restriction. However, a lower Pb sensitivity was seen for both species throughout generations. Both species also showed that under food restriction neonates' sizes were larger than those kept under regular food, while reproduction was considerably reduced. Food restriction also generated opposite outcomes on both species, such as D. magna epigenetic changes and D. similis phenotypic acclimation to Pb. Besides, D. magna pre-exposed to Pb presented lower sensitivity to mancozeb, while the contrary was shown by D. similis. This study indicates that daphnids are capable of acquiring a lower sensitivity to Pb across a long-term exposure, and that Pb pre-exposure can affect the sensitivity to other chemicals. Also, different patterns in multi-generational responses from monophyletic species (especially under oligotrophic media, common on natural habitats) acknowledge the use of representative or native species to assess the effect of contaminants, since monophyletic species can provide different toxicity outputs.

Effects of hydrochemistry variables on the half-life of mancozeb and on the hazard index associated to the sum of mancozeb and ethylenethiourea.

Mancozeb is a dithiocarbamate non-systemic agricultural fungicide with multi-site, protective action. It helps to control many fungal diseases in a wide range of field crops, fruits, nuts, vegetables, and ornamental plants. We have investigated the stability profiles of mancozeb in aqueous solutions to determine the effect of pH, temperature and light on the degradation process of mancozeb. In addition, the toxicological risk for humans associated with the joint intake of mancoze7b and its final degradation product, ethylenethiourea (ETU), was calculated and modelled as a function of the experimental conditions. Stability study results showed a very low stability profile of mancozeb in all the aqueous solutions with rapid degradation that varied with experimental conditions. The process followed first order kinetics. The study of the degradation kinetics showed a significant effect of pH*temperature interaction on the degradation process. The results also expressed that light has a greater impact on the stability of mancozeb and the formation of ETU. The current study concludes that mancozeb is unstable in aqueous solutions, particularly at an acid pH, in addition to presenting both severe light and lower temperature sensitivity. The toxicological risk associated with mancozeb degradation increases with time and temperature, being higher at basic pH and in absence of light.

Phytoremediation of groundwater contaminated with pesticides using short-rotation willow crops: A case study of an apple orchard.

The occurrence of pesticides in groundwater represents an important health issue, notably for population whose drinking water supply source is located in agricultural areas. However, few solutions have been considered with regard to this issue. We tested the efficacy of a vegetal filtering system made of shrub willows planted at a high density (16,000 plants ha(-1)) to filter or degrade pesticides found in the groundwater flowing out of an apple orchard. Ethylene urea (EU), ethylene thiourea (ETU), tetrahydrophthalimide (THPI), atrazine, and desethylatrazine were monitored in the soil solution in willow and control plots over one growing season. ETU and atrazine concentrations were lower in the willow plots relative to the control plots, whereas desethylatrazine concentration was higher in the willow plots. No significant difference was detected for EU and THPI. Furthermore, pesticide concentrations displayed complex temporal patterns. These results suggest that willow filter systems can filter or degrade pesticides, notably ETU and atrazine, and could be used for phytoremediation purposes. Yet, this potential remains to be quantified with further studies using experimental settings allowing more estimation in time and space.

Thiol-reactivity of the fungicide maneb.

Maneb (MB) is a manganese-containing ethylene bis-dithiocarbamate fungicide that is implicated as an environmental risk factor for Parkinson's disease, especially in combination with paraquat (PQ). Dithiocarbamates inhibit aldehyde dehydrogenases, but the relationship of this to the combined toxicity of MB + PQ is unclear because PQ is an oxidant and MB activates Nrf2 and increases cellular GSH without apparent oxidative stress. The present research investigated the direct reactivity of MB with protein thiols using recombinant thioredoxin-1 (Trx1) as a model protein. The results show that MB causes stoichiometric loss of protein thiols, reversibly dimerizes the protein and inhibits its enzymatic activity. MB reacted at similar rates with low-molecular weight, thiol-containing chemicals. Together, the data suggest that MB can potentiate neurotoxicity of multiple agents by disrupting protein thiol functions in a manner analogous to that caused by oxidative stress, but without GSH depletion.

SUBCHRONIC MANCOZEB TREATEMENT INDUCED LIVER TOXICITY VIA OXIDATIVE STRESS IN MALE WISTAR RATS.

Mancozeb is a manganese/zinc ethylene-bis-dithiocarbamate fungicide that is widely used in agriculture to control a broad variety of fungal infections of both vegetables and ornamental plants. The present study has been carried out to investigate the possible effect of mancozeb on animal the oxidative stress and some of the biochemical markers in male Wistar rats. In this experiment, adult male rats weighing between 200 and 250 g were treated per os for 4 weeks with two different doses of 800 and 1200 mg/kg per day. Reduced glutathione (GSH) levels were decreased in all treated groups compared to control ones. It has been observed a significant increase in the fresh weight of liver in individuals of both doses. Moreover, mancozeb exposure caused a significant (p < 0.05) fall in aspartic aminotransferase (AST) and alanine aminotransferase (ALT) in group treated with 1200 and 800 mg/kg/day. Similarly, alkaline phosphatase (ALP) activity underwent a significant (p < 0.05) increase in both groups. The obtained observations clearly reveal hepatotoxic effects of mancozeb in rats and constitute, therefore, an environmental health risks to living organisms.

Mancozeb-induced behavioral deficits precede structural neural degeneration.

Manganese-containing fungicides like Mancozeb have been associated with neurodegenerative conditions like Parkinson's disease. We examined the behavioral damage and differential neuronal vulnerability resulting from Mancozeb exposure using Caenorhabditis elegans, an important mid-trophic level soil organism that is also a powerful model for studying mechanisms of environmental pollutant-induced neurodegenerative disease. The dopamine-mediated swim to crawl locomotory transition behavior is exquisitely vulnerable to Mancozeb, with functional impairment preceding markers of neuronal structural damage. The damage is partially rescued in mutants lacking the divalent metal transporter, SMF-1, demonstrating that some, but not all, of the damage is mediated by manganese. Increasing concentrations of Mancozeb recruit additional behavioral dysfunction, notably serotonin-mediated egg-laying behavior, but without evident serotonergic neuronal structural damage. Thus, measurements of behavioral dysfunction are a sensitive early marker of fungicide toxicity that could be exploited to examine further mechanisms of neuron damage and possible therapeutic interventions. These results also provide important insight into the consequences of fungicide use on the ecological behavior of nematodes.

Toxicity of a mancozeb containing formulation and Cd-sulphate to chicken embryos after administration as single compounds or in combination.

Environmental pollution of metal modelled by cadmium-sulphate and a 80% mancozeb containing fungicide formulation (Dithane M-45) were studied on chicken embryos after administration as a single compounds or in combination. The test materials were injected into the air-chamber in a volume of 0.1 ml/egg on day 0 of incubation. The concentration of cadmium-sulphate was 0.01%. The applied concentration of Dithane M-45 fungicide was 0.2%. Evaluation was done on day 19 of the hatching period. The individual administration of cadmium-sulphate and the 80% mancozeb containing fungicide formulation did not cause a significant reduction in body weight as compared to the control data. Embryonic mortality increased at all individual treated groups and reached almost a 35% rate. After the individual administration of pesticide, the number of chicken embryos with developmental anomalies did not differ markedly from the control. After the combined administration of cadmium-sulphate and the 80% mancozeb containing fungicide formulation (Dithane M-45) on day 0 of the hatching period embryonic mortality markedly increased. 88% of the treated embryos were dead. Results from the combined administration of cadmium-sulphate and an 80% mancozeb containing fungicide formulation (Dithane M-45) caused higher embryomortality with respect to individual toxicity test of cadmium-sulphate and fungicide in our study.

Genotoxic and mono-oxygenase system effects of the fungicide maneb.

The in vivo effects of a commercial preparation of maneb on mono-oxygenase activities of hepatic microsomes of basal and induced rats were examined. In vitro experiments with the D7 strain of yeast Saccharomyces cerevisiae were also performed. In both basal and induced rats maneb caused a decrease in cytochrome P-450 content and aniline hydroxylase. Immunoblotting analysis using anti-P-450 IIE1 antibodies confirmed the data obtained for aniline hydroxylase activity. Maneb was toxic in cells of S. cerevisiae. On the basis of in vivo and in vitro experiments it can be concluded that maneb possesses a toxic activity attributable to its main metabolite ethylene thiourea. Immunoblotting analysis indicates that maneb biotransformation influences the IIEI P-450 isoform.

Biochemical alterations in Bradyrhizobium sp USDA 3187 induced by the fungicide Mancozeb.

We have previously shown that fungicide Mancozeb causes a 50% decrease in Bradyhizobium sp USDA 3187 growth rate and affects the bacteria-root symbiotic interaction. In order to elucidate the fungicide toxicity mechanism we determined the effects of Mancozeb on cell chemical composition, glutathione (GSH) content (molecule involved in the detoxification process), glutathione S-transferase (GST) activity and on polyamine, exopolysaccharides, capsular polysaccharides and lipopolysaccharides. Mancozeb produced biochemical alterations in membrane composition, polysaccharides and polyamines. In spite of the increment of GSH content and GST activity, they are not enough to prevent the growth diminution.

Environmental and biological monitoring of exposure to mancozeb, ethylenethiourea, and dimethoate during industrial formulation.

The results of environmental (11 subjects) and biological (57 subjects) monitoring of exposure to mancozeb, ethylenethiourea (ETU), and dimethoate are reported for employees of a firm producing commercial formulations containing these active ingredients. Urinary excretion [GM(GSD)] of ETU (microg/g creatinine) and alkylphosphates [dimethylphosphate (DMP) + dimethylthiophosphate (DMTP) + dimethyldithiophosphate (DMDTP)] (nmol/g creatinine) was 65.3(4.8) and 419.2(2.1), respectively, for employees engaged in the formulation of a product containing 80% mancozeb (n = 9), 36.6(1.9) and 296.4(2.4) for those formulating a product containing 35% mancozeb (n = 9), 9.5(6.1) and 1022.4(3.0) for those engaged in plant maintenance and internal transport of materials (n = 6), 10.3(4.2) and 322.8(3.3) for those engaged in packaging the mancozeb formulations (n = 16), 4.4(3.3) and 2545.4(3.9) for those formulating a product containing 40% dimethoate (n = 11), and 3.0(2.7) and 871.7(3.3) for those bottling the same dimethoate formulation (n = 10). Air concentrations (microg/m3) ranged from 25.3 to 194.4 for dimethoate, from 0.2 to 1.3 for ETU, and from 139.9 to 949.0 for mancozeb. Urinary excretion of ETU and alkylphosphates showed a significant correlation with mancozeb (r2 = .971), and ETU (r2 = .858), and dimethoate (r2 = .955) contamination of the hands. Potential dose estimates showed that the potential respiratory doses of mancozeb and dimethoate accounted, on the average, for 38% of the total potential dose. The potential respiratory dose of ETU was 7% of the total potential dose. Total estimated absorption did not exceed the accepted daily dose (ADI) for ETU and mancozeb, but the ADI for dimethoate was exceeded. Serum and erythrocyte cholinesterase activities in workers formulating dimethoate products were not significantly different before and after exposure.

[Evaluation of urinary excretion of ethylenethiourea in subjects occupationally and non-occupationally exposed to ethylenebis(dithiocarbamates)]. / Valutazione dell'escrezione urinaria di etilentiourea in soggetti professionalmente e non professionalmente esposti ad etilenbisditiocarbammati.

Ethylenebisdithiorcabamate (EBDC) fungicides are broken down metabolically and in the environment to ethylenethiourea (ETU), a suspected carcinogen. Urinary ETU was assayed in control groups and subjects occupationaly exposed to EBDC and was found to be an excellent indicator of exposure both to ETU and EBDC. Correct use of protective clothing greatly reduced exposure and urinary excretion of ETU. ETU was excreted, albeit in low concentrations, in a high percentage (91% and 30%) of subjects in both control groups, demonstrating its utility as an indicator of widespread EBDC contamination.

Ethylenethiourea in air and in urine as an indicator of exposure to ethylenebisdithiocarbamate fungicides.

Ethylenethiourea (ETU) is a ubiquitous impurity of the ethylenebisdithiocarbamate (EBDC) fungicides widely used in agriculture and forestry. In the present study, ETU was used as a measure of the exposure to EBDC on potato farms and in pine nurseries during the application of EBDC fungicides and the weeding of the sprayed vegetation. Biological and hygienic monitoring was carried out through the analysis of ETU in the breathing zone and the urine of exposed workers. Even if the concentrations of ETU in the ambient air of pine nurseries exceeded those of potato farms, the concentrations of ETU in the urine of potato farmers exceeded those of pine nursery workers. This result may have been due better protective equipment in the pine nurseries. The excretion rate was 6-10 ng/h during the first 60 h after the cessation of exposure, and it diminished thereafter to 0.2 ng/h over a 22-d observation period.

Examination of the interaction of decis and dithane in rats.

Acute (LD50) and short-term (14 days) toxicological examinations were performed in animal experiments on the interaction of a synthetic pyrethroid Decis 2,5 EC (25 g deltamethrin/l) and of ethylene-bisdithiocarbamate/Dithane M-45 (80% mancozeb), using a 1:5 deltamethrin/mancozeb mixture. LD50 value of the mixture was similar to that of the more toxic Decis. In the short-term examination, some pathologically high AST and ALT values were observed in the treated groups and the deltamethrin content of fatty tissue samples increased parallel with the increase of Decis consumption. The chymotrypsin and lipase activities in the small intestinal mucosa and gamma-GT and LAP activities in the content of the bowels were reduced in several treated groups. The administration of Dithane in a dose in accordance with 20% of the LD50 value (3125 mg/kg b.m.) proved to be more toxic than expected and caused the death of the animals.

The response of Fusarium oxysporum to dithane M-45 and Pseudomonas aeruginosa.

The interaction of the fungicide Dithane M-45 (active ingredient mancozeb), with Pseudomonas aeruginosa strain not sensitive to the compound, was studied. Pseudomonas aeruginosa produced a fungicidal compound inhibiting the growth of Fusarium oxysporum. Simultaneously Pseudomonas aeruginosa degraded the EBIS (ethyleneisothiocyaniane) formed from the spontaneous decomposition of the fungicide which is highly toxic for Fusarium oxysporum. When the fungicide was used as seed dressing together with the bacteria the inhibition of the fungus was reduced to the interaction of both fungicidal compounds.

Allergic contact dermatitis from ethylene thiourea.

A case of allergic contact dermatitis to the rubber additive material ethylene thiourea (ETU) is presented. Testing for cross-sensitivity gave a positive reaction to the fungicide Maneb, manganous ethylenebisdithiocarbamate, but this reaction was probably due to ETU which was detected in Maneb by thin-layer chromatography. Different sources of ETU are reported.